DK171069B1 - Preparation which comprises a lanthionine-containing bacteriocin in combination with a chelating agent and/or a surface-active agent - Google Patents

Abstract

Compositions comprising a lanthionine-containing bacteriocin such as nisin and a surfactant, especially a nonionic one, are useful as bactericides, especially against Gram positive bacteria. They have both food and medicinal applications. To avoid overlap of Claim with the parent application, chelating agents are excluded from the claimed compositions and uses.

Description

DK 171069 B1

The invention relates to a composition containing a lanthionine-containing bacteriocin in combination with a chelating and / or surfactant.

Nisin is a polypeptide with antimicrobial properties, produced in nature by various strains of the bacterium Streptococcus lactis. Nisin is a known food preservative that inhibits the growth of spores of certain species of gram-positive bacteria.

Although nisin is sometimes mistakenly and imprecisely referred to as an antibiotic, this substance is more correctly classified as a bacteriocin, ie. a proteinaceous substance produced by bacteria and having only antibacterial activity against species closely related to the origin of the nisin. Nisin is a naturally occurring preservative found in low concentration in milk and cheese, and this substance is believed to be completely non-toxic and non-allergenic to humans.

Nisin has recently been recognized as safe by the FDA as a direct food ingredient in pasteurized 20 cheese, pasteurized processed cheese and pasteurized or pasteurized processed cheese with fruits, vegetables or meat. Furthermore, since nisin is a polypeptide, any nisin residues remaining in foods will be digested rapidly.

25 A summary of the properties of nisin can be found in A.

Hurst, Advances in Applied Microbiology 27., 85-123 (1981).

This publication describes what is generally known about nisin. Nisin produced by Streptococcus lactis is commercially available as an impure preparation, Nisaplin®, available from Aplin & Barrett Ltd., Dorset, England, and can be obtained by isolating naturally occurring nisin from cultures of Streptococcus lactis upon which the nisin concentrated using known methods. Methods for producing nisin have also been described using altered 35 strains of Streptococcus, cf. U.S. Patent No. 4,716,115. It should also be possible to produce nisin using recombinant DNA technology 2 DK 171069 B1.

Nisin has been used effectively as a preservative in dairy products, e.g. processed cheese, cream and milk. The use of nisin in processed cheese products is the subject of recently issued patents, cf. U.S. Patent Nos. 4,584,199 and 4,597,972. The use of nisin to inhibit the growth of certain gram-positive bacteria is well documented. The complete success and recognition of nisin as a food preservative has so far been hampered by the assumption that nisin is ineffective against gram-negative and many gram-positive bacteria. Gram-negative bacteria are practically always present in association with Gram-positive bacteria and are a major source of food destruction and contamination, cf. U.S. Patent No. 4,584,199 and U.S. Patent No. 4,597,972, and Tsai and Sandine, "Conjugal Transfer of Nisin Plasmid Genes from Streptococcus Lactis 7962 to Leuconostoc Dextranicum 181, Applied and Environmental Microbiology, February 1987, page 352," A Natural Preservative ", Food Engineering 20 International, May 1987, pages 37-38 and "Focus on Nisin", Food Manufacture, March 1987, page 63.

Contrary to the prior art, it has been found that preparations containing nisin and other lanthionine-containing bacteriocins in combination with various non-bactericidal agents have increased or enhanced broad spectrum bactericidal activity against gram-negative bacteria as well as increased activity against a wider spectrum of gram-positive bacteria than nisin or the other bacteriocins alone. The increased bactericidal activity against gram-positive bacteria is present in a pH range that is wider than previously thought. The invention thus relates to a composition characterized in that it contains a lanthionine-containing bacteriocin in combination with a chelating agent and / or surfactant. A number of preferred embodiments 35 of the composition of the invention are set forth in claims 2-12.

More specifically, it has been found that a solution of approx. 0.1 µg of nisin per ml to approx. 300 µg of nisin per ml in the presence of from ca. 0.1 mM to approx. 20 mM of a chelating agent, e.g. EDTA, in practice, eliminates the growth of gram-negative bacteria, e.g. Salmonella typhimurium, 5 Escherichia coli, Pseudomonas aeruginosa, Bacterioides gin givalis, Actinobacillus actinomycetescomitans and Klebsiella pneumoniae, and are more active against gram-positive bacteria, e.g. Staphylococcus aureus, Streptococcus mutans, Listeria monocytogenes, Streptococcus agalactiae and Coryneform bacteria, than nisin alone. Although the increase in nisin activity with the chelating agent is concentration dependent, concentrations of EDTA beyond 20 mM are contrary to expectations inhibiting the bactericidal activity of nisin. In the presence of a proteinaceous carrier and polyvalent monomers, e.g. however, serum albumin, collagen, gelatin, casein and keratin, the inhibition of nisin at concentrations of EDTA greater than 20 mM, however, significantly reduces the extent of the useful range for EDTA enhancement of nisin activity.

20 It has also been found that a solution of from ca.

0.1 µg of nisin per ml to approx. 300 µg / ml and from approx. 0.1 mM to approx. 20 mM of a chelating agent will further enhance the efficacy of nisin against gram-negative and gram-positive bacteria in the presence of from ca. 0.01 to approx. 1.0% surfactant. It has further been found that nisin in the presence of surfactant alone has increased activity against gram-positive bacteria.

According to the present invention, suitable chelating agents include EDTA, CaEDTA, CaNa2EDTA and other alkyl diamine tetraacetates, EGTA and citrate. Surfactants which are valuable as detergents and which are suitable for combination with nisin, with or without EDTA, include, for example. the nonionic surfactants Tweens, Tritons and glycerides; ionic surfactants, e.g. fatty acids, 35 quaternary compounds, anionic surfactants, e.g. sodium dodecyl sulfate, and amphoteric surfactants, e.g. cocamidopropyl betaine, and emulsifiers.

Since gram-positive and gram-negative bacteria are almost always found together in food, the effectiveness of nisin preparations against gram-negative bacteria such as Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacterioides gin-givalis and Actinobacillus actinomycetus Other gram-negative pathogens and gram-positive bacteria are of great use. The bactericides are particularly useful for controlling and preventing contamination of raw ingredients, processed foods and beverages associated with bacterial pathogens and other microbial, destructive organisms. Potential food-related uses include the processing of meat products, in particular poultry, eggs, cheese and fish 15, as well as processing of food packaging materials and handling equipment. Additional uses include food preservation, e.g. in processed cheese, cream, milk, dairy products and in the purification of poultry, fish, meat, vegetables and dairy and food processing app-20. The use of the nisin preparations should not be limited to food-related applications, and the nisin preparations can be used in any situation where there is a need or desire to eliminate gram-negative and gram-positive bacteria.

The compositions may be dissolved in a suitable carrier, e.g.

an aqueous solvent or buffer, or suspended in any suitable liquid, colloid or polymeric matrix to form bactericides. The compositions or bactericides may be incorporated into ointments or coatings for medical use, e.g. treatment of infections, wound dressings or surgical implants and as broad-spectrum disinfectants for rinsing the skin or oral rinsing, disinfectants, wipes or lotions. The bactericides can be used for the purification of 35 medical instruments, in preoperative surgical cleansers and the like. The bactericides are particularly useful in circumstances where a disinfection of the environment is desired, but where chemical germicides are excluded due to the risk of corrosive or otherwise toxic residues.

In contrast to the activity of most broad-spectrum germicides which are adversely affected by the presence of complex organic matter, the compositions of the invention act as bactericides in the presence of organic matter, e.g. milk or serum.

It is known that nisin optimally inhibits the growth of a few closely related gram-positive bacteria, in particular certain gram-positive spore-forming bacteria at a pH of 5.0. The bactericidal activity of nisin in solution with a chelating agent is surprisingly rapid and greatly enhanced against a wide range of gram-positive bacteria at pH values greater than pH = 5.0, and is further activated against gram-negative bacteria at both acidic and basic pH values, preferably in the pH range of 5.0 to 8.0. This unexpectedly rapid and broad-spectrum bactericidal activity of chelating agent-activated nisin makes this suitable for use as, among other things, a disinfectant.

Nisin belongs to the class of peptide bacteriocins containing lanthionine. Also included in this class are subtilin, epidermin, cinnamycin, duramycin, ancovenin and Pep 5. These bacteriocin peptides are individually produced by 25 different microorganisms. Subtilin obtained from certain cultures of Bacillus subtilis and epidermin obtained from certain cultures of Staphylococcus epidermidis have been found to have molecular structures very similar to the molecular structure of nisin, cf. Hurst, pages 85-86, and Schnell et al., 30 Nature 333 276-278. Therefore, it is believed and has actually been shown that, due to the molecular similarities, other lanthionine-containing peptide bacteriocins are as effective as nisin in combination with chelating agents and / or nonionic surfactants for the elimination of gram-negative and gram-positive bacterial contaminants.

The efficacy of nisin and other lanthionine-containing peptide-bacteriocin preparations as bactericides against gram-negative bacteria is surprising, since the prior art generally omits this activity for nisin. The increased activity of nisin against gram-positive bacteria 5 in the presence of EDTA at a pH of more than 5.0 is unexpected, since hitherto it has been thought that nisin activity is optimal at a pH of 5.0 . Furthermore, the recognition of such efficacy of the nisin- and lanthionine-containing peptide bacteriocin preparations as bactericides satisfies a long-standing need in the science of food preservation, which has suffered from the lack of an acceptable, natural, non -toxic agent that works against a wide range of bacteria.

To detect the superior and unexpectedly rapid activity of a preparation containing nisin, EDTA and / or various surfactants against both gram-negative and gram-positive bacteria, a number of experiments have been carried out with the bactericides. These experiments serve to illustrate the invention. Other lanthionine-containing peptide bacteriocins 20 are effective substitutes for nisin, and chelating agents other than EDTA are effective substitutes for EDTA, cf. the following experimental results.

All experiments in the following examples are performed at 37 ° C. The efficacy of the enhanced broad-spectrum bactericides is determined by determining the bactericidal activity as measured as the percent bacterial survival after treatment with the bactericide. Generally, bacteria are collected by centrifugation for 2 minutes after incubating a cell suspension at 107 cells per ml. ml of the species to be examined with the novel bactericide for a specified period of time. The resulting bacterial pellet is washed free of the bactericide with an auxiliary buffer, referred to herein as a phage buffer (50 mM tris-HCl buffer having a pH of 7.8, 1 mM MgSC> 4.4 mM CaCl 1 M NaCl and 0.1% gelatin) are resuspended and serially diluted in phage buffer, and 100 ml of the suspended bacteria is spread on nutrient agar plates.

7 DK 171069 B1

Surviving bacteria are determined as colony-forming units (CFUs) after incubation for 24-48 hours at 37 ° C. An effective bactericide of the invention is one which allows less than 0.1% of the original viable number of bacteria to survive.

Example 1

Activity of Nisin and a Chelating Agent against Gram-Negative Bacteria (S. typhimurium) 10 As can be seen from the following Table I, two experiments were performed in 20 mM Tris, pH = 8.0, at 37 ° C by demonstrating the effect of the bactericide containing nisin and the chelating agent EDTA alone. Experiment # 1, a control experiment, is performed without EDTA and shows the effect of nisin alone against the gram-negative bacterium S. thyphimurium. The increased concentrations of nisin exhibit some activity, but even the activity of higher concentrations in the presence of EDTA, which gives 1.6% survival at 100 μg of nisin per day. ml is quite insufficient for a nutrient preservative. The level of bactericidal activity achieved with nisin and EDTA is significant.

Table i

Originally 25 viable

Test Bacterial EDTA Nisin (µg / ml) No. of Quantities (mM) 0 10 30 50 100 300

Percent S. typhimurium survival after 3 hours 1 3.0 x 106 0 100 51.3 - 7.0 1.6 2 5.7 x 106 20 2.5 - 10 ~ 3 - <10'4 <10 " 4

Trial # 2, cf. Table I, is performed using 35 of nisin plus 20 mM EDTA and shows the surprising activity of the nisin preparation to eliminate the gram-negative bacterium studied.

It can be seen from Table I that in experiment # 2 in an 8 DK 171069 B1 concentration of 20 mM EDTA and 30 μg of nisin per ml, the bactericide has a marked bactericidal activity against S. typhimurium, while at a nisin concentration of 100 μg / ml and the higher nisin and EDTA bactericide practically eliminates the bacteria (percent survival less than 10'4, which indicates that no surviving bacteria are present at the trial). Thus, the combination of EDTA and nisin exhibits a potentiated activity that is more than 1000 times the activity of nisin alone.

The following Table IA shows the activity of subtilin and chelating agent against the gram-negative bacterium Salmonella typhimurium.

Table IA

Activity of Subtilin and Chelating Agent against the Gram-Negative Bacteria Salmonella Typhimurium

Carrier Subtilin EDTA rrM

(/ xg / ml) 0 0.1 1 3 10 100 20% survival 2 hours 0 100a

Buffer pH 8.0b 5 5 0.008 0.0004 0.004 0.04 11.3 25

Buffer pH 5.0C 5 0.06 0.04 0.0003 0.00030.08 6 30 a Initially Viable Bacterial Number 3 x 10 4 CFQ / ml b 20 nM Tris-HCl, pH 8.0 c 50 rrM Na-Acetate , pH 5.0

Example 2 Activity of Nisin, a Chelating Agent, and a Surfactant against Gram-Negative Bacteria (S. typhimurium)

Four tests are performed, cf. Table II, to determine the effect on S. typhimurium of the bactericide containing nisin and both EDTA and the surfactant 40 Triton X-100 in 20 mM tris, pH = 8.0, at 37 ° C. The control, 9 DK 171069 B1, cf. Experiment # 1, is identical to the control in Example 1, cf. Table I.

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11 DK 171069 B1

Trial # 2, cf. Table II, performed using nisin and 1.0% Triton X-100, but without EDTA. The presence of the detergent alone inhibits the activity of nisin against the gram-negative bacterium and nisin is inactive. For trials 5, no. 3 and no. 4, cf. however, Table II representing the present invention represents the presence of 20 mM EDTA in combination with Triton X-100, a bactericide that appreciably increases the bactericidal activity of nisin against S. typhimurium. Combination of Triton X-100 with EDTA, but 10 without nisin, is actually effective, but to a lesser extent than in the presence of nisin. Albeit in both Experiments Nos. 3 and 4, cf. Table II, the nisin combinations are very effective, is the concentration of 1.0% Triton X-100, cf. Experiment # 4, Table II, most effective.

The presence of the nonionic surfactant, Triton X-100, in combination with EDTA increases even the activity of nisin against gram-negative bacteria even more than the bactericide containing nisin and EDTA alone, cf. Example 1.

The following Tables IIA-IID show the activity of nisin and various chelating agents against E. coli and S. mutans.

Table IIA

25 Activity of nisin and chelating agents against Escherichia coli

Chelating agent 1 mM ^

Nisin (/ zg / ml) 0 EDTA DTPA CDTA EGTA 1 0% survival 1 minute 100 100a 0.61 4.2 4.3 10.9 3 5 a initially viable bacterial count 1 x 107 CFU / ml b 10 mM Tris-HCl , pH 7.3 40 12 DK 171069 B1

Table IIB

Activity of nisin and CaNa2EDTA against Streptococcus mutans 5

CaNa2EDTA mM

Carrier Nisin (µg / ml) 0 1 10% survival 1 minute 10% FCSb 3.0 3.0 0.052 15

Initially viable bacterial count 3 x 106 CFU / ml b 10% fetal calf serum 20

Table IIC 25

Activity of nisin and citrate against Streptococcus mutans 30

Carrier Nisin Citrate trM

(μς / τύ.) 0 0.1 1 3 10 100 35% survival 2 hours

Buffer pH 7.3b 0.3 3.65 1.25 0.1 0.42 0.2 0.35 40

Initially viable battery count 3 x 106 CFU / ml b 10 mM Tris-HCl, pH 7.3 45 13 DK 171069 B1

Table IIP

Activity of nisin and citrate against Escherichia coli 5 Carrier Nisin% Citrate (μg / τcl) 0 0.1 0.3 1 3% survival 1 minute 10

Buffer pH 3.5 0 100 100 56 73 100 100 100 0.0025 0.0006 0.56 14.6 15

Initial Viable Battery Number 5 x 107 CFU / ml Example 3 20 Activity of Nisin, a Chelating Agent and a Surfactant against Gram-Negative Bacteria (S. typhimurium)

Table III shows the increased activity against S. typhimurium of the bactericide containing nisin, 20 mM of the chelating agent EDTA and the nonionic surfactant 25 Tween 20 in 20 mM tris, pH = 8.0, at 37 ° C. As with the Triton X-100, cf. Example 2, the combination of nisin and EDTA with (1%) Tween 20 is most effective.

14 DK 171069 B1

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DK 171069 B1

The following Tables IIIA and HIB show the activity of nisin combined with a chelating agent and various surfactants.

5 Table IIIA

Activity of nisin and nonionic surfactants against Escherichia coli 10 Tween products Carrier Nisin [μg / ml} 0 Tw20 Tw40 Tw80

+ 1 mM EDTA

15% survival 1 minute 0 100a 100 100 100

Buffer pH 7.0b 3 0.25 0.001 0.007 0.007 2 0 _ a Initially viable bacterial count 1 x 107 CFU / ml b 20 mM Tris-HCl, pH 7.0

Table HIB

30 Activity of nisin and amphoteric surfactant against Streptococcus mutans% cocamidopropylbetaine Carrier Nisin (μg / ml) 0.1

35 + 1 mM EDTA

% survival 1 minute 40 10% FCSb 0 100 11 3 30 0.07 a original viable bacterial count 1 x 107 CFU / ml 45 b 10% fetal calf serum 16 DK 171069 B1

Example 4

Activity of nisin, a chelating agent and a surfactant against gram-negative bacteria (Escherichia coli)

The effect of the bactericide containing nisin and EDTA against the gram-negative bacterium E. coli is demonstrated as shown in the following Table IV.

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18 DK 171069 B1

The experiments with and without EDTA are performed in 20 mM Tris-buffer solution, pH = 8.0, at 37 ° C, with an initially viable bacterial count of 1 x 10 7 E. coli cells per ml. The effects of the bactericide are measured as a function of the 5 percent bacterial survival after 2 hours.

In trial # 1, cf. control, Table IV, without EDTA, nisin exhibits little meaningful activity to eliminate E. coli. In trial # 2, cf. however, in Table IV, where 20 mM EDTA is present, the bactericidal preparation 10 exhibits significant activity relative to the E. coli bacteria. The activity increases in efficiency as the concentration of nisin is increased. The combination of nisin with EDTA as a bactericide shows a 100-fold potent increase in activity against E. coli. In trial # 3 and no.

15 4, cf. Table IV, it can be seen that Triton X-100 has no potent bactericidal activity against E. coli. In fact, Triton X-100 appears to inhibit nisin activity against gram-negative bacteria, as found with S. typhimurium, cf. Table II. However, the overall increase in nisin-20 activity with EDTA substantially abolishes the inhibitory effects of Triton X-100, as shown in Tables II and IV.

Thus, it appears that the bactericide containing nisin and a chelating agent, e.g. EDTA, is an effective food preservative against various types of gram-negative bacteria, even in the presence of surfactants.

Example 5 30 Activity of Nisin and a Chelating Agent against Gram-Negative Bacteria (Klebsiella pneumoniae)

The effect of the bactericide containing nisin and EDTA alone on the gram-negative bacterium K. pneumoniae is demonstrated as shown in Table V.

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DK 171069 B1

The two experiments, one with and one without EDTA (control), were performed in 20 mM Tris buffer, pH = 8.0, at 37 ° C, with an initially viable bacterial count of 107 cells of K. pneumoniae per ml. The efficacy is measured as a function of the percentage 5 bacterial survival after 2 hours.

In trial # 1, cf. control, Table V, without EDTA, nisin exhibits little meaningful bactericidal activity against K. pneumoniae. In trial # 2, cf. however, in Table V, where 20 mM EDTA is present, the bactericide exhibits a significant activity against K. pneumoniae. The activity increases in efficiency as the concentration of nisin is increased.

Example 6

Nisin activity against gram-negative bacteria (Salmonella typhimurium) is dependent on the concentration of chelating agent

The data presented in Table VI shows that the increased activation of nisin against gram-negative bacteria (S. typhimurium) depends on the concentration of EDTA in either 50 mM 20 sodium acetate, pH = 5.0, or in 20 mM tris, pH = 8.0, at 37 ° C. 1 21 DK 171069 B1 o m o o i i ^ ro

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For trial # 1 and # 3, cf. the controls, Table VI, where EDTA concentrations of up to 100 mM without nisin are used, little meaningful activity against S. typhimurium is observed at either pH = 5.0 (no. 1) or pH = 5 8.0 (no. 3). In Experiments Nos. 2 and 4, cf. Table VI, where 100 µg of nisin per However, in combination with EDTA, the bactericides exhibit significant activity against S. typhimurium. The activity of the bactericides is of the same magnitude both at an acidic pH (5.0) 10 and at a basic pH (8.0), despite the fact that the activity of nisin alone against gram-positive bacteria is optimal at a pH of 5.0.

The increase in nisin activity with EDTA is concentration dependent, being optimal in the range of 0.2 mM 15 to 10 mM at the pH values of 5.0 and 8.0. At concentrations higher than 10 mM EDTA, the increase in nisin activity with EDTA is surprisingly diminished. The decrease in activation is significantly greater at a pH of 8.0 than at a pH of 5.0.

20

Example 7

Nisin and a chelating agent against gram-negative bacteria (S. typhimurium)

The increase of nisin activity by EDTA against gram-25 negative bacteria in the presence of biological tissue is detected with S. typhimurium on chicken muscles and is shown in the following Table VII.

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Incubations are performed in either 50 mM sodium acetate, pH = 5.0, or in 20 mM tris, pH = 8.0, at 37 ° C.

Chicken muscle cubes are rinsed with sodium hypochlorite and povidone iodine prior to use. For grafting the 5 tissue, the cubes of chicken muscle are dipped into a cell suspension with 10® cells of S. typhimurium per ml. ml in 20 mM tris-HCl, pH = 8.0. Excess moisture is removed from the dipped cubes upon drainage. The chicken samples are placed in sufficient buffer containing the nisin preparation to cover the tissue and incubated for 2 hours at 37 ° C, after which the tissue is removed to a sufficient amount of phage buffer to cover the tissue. The bacteria that remain in the test solution are collected by centrifugation, washed with phage buffer and combined with bacteria washed from 15 tissue with phage buffer. The combined samples, termed "non-adherent" cells, are serially diluted and aliquots of 100 μΐ are plated by the surviving bacteria.

For trial # 1 and # 3, cf. Table VII, in the absence of nisin at a pH of either 5 or 8, EDTA 20 alone has no significant effect on the survival of S. typhimurium. For trial # 2 and # 4, cf. Table VII, where there are 300 µg of nisin per however, the bactericides exhibit significant activity against S. typhimurium on chicken muscle at both pH = 5.0 and pH = 8.0.

The assay of nisin activity with EDTA is concentration dependent, the optimum concentration being in the range of 0.3 to 10 mM EDTA at both pH values, 5.0 and 8.0. At concentrations higher than 10 mM EDTA at pH = 8.0, the activation of nisin with EDTA is decreased. However, as can be seen from Experiment 5, cf. Table VII, however, the efficacy of nisin against S. typhimurium on chicken muscle is expressed throughout the range of EDTA concentrations up to 100 mM in the presence of 1.0% bovine serum albumin at a pH of 8.0.

Bactericides containing nisin and low concentrations of chelating agent, e.g. EDTA, in the range of 0.1 to 20 mM, can thus be highly effective in eliminating or preventing contamination of foods with gram-negative bacteria.

Example 8

Titration of nisin activity against gram-negative bacteria (S. tyhphimurium)

At the optimum concentration of chelating agent, the effectiveness of the bactericide in tris buffer against 10 gram negative bacteria is shown to be significant, as shown in Table VIII.

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As shown in Experiment 2 of Table VIII, as little as 0.3 μg of nisin per ml of 1.0 mM EDTA in 20 mM tris at pH = 8.0 in the presence of 1% bovine serum albumin (BSA) significantly decreases the survival of S. typhimurium. The bactericide is as active against gram-negative bacteria as nisin alone is active against gram-positive streptococci.

Example 9

Titration of nisin activity against gram-negative bacteria 10 (S. typhimurium)

At the optimum concentration of the chelating agent, the efficacy of a bactericide against gram-negative bacteria in the presence of S. typhimurium biological tissue on chicken muscle is demonstrated, and the results are shown in Table IX.

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Chicken muscle cubes are rinsed with sodium hypochlorite and povidone iodine prior to use. For grafting the tissue, cubes of chicken muscle are dipped into a cell suspension containing 10® cells of S. typhimurium per ml. ml in 5 mM Tris-HCl, pH = 8.0. Excess moisture is removed from the dipped cubes upon drainage. The tissue is placed in sufficient buffer containing the nisin preparations to cover the tissue and incubated for 2 hours at 37 ° C, then the tissue is transferred to a sufficient amount of phage buffer to cover the tissue. The bacteria that remain in the test solution are collected by centrifugation, washed with phage buffer and combined with bacteria washed out of the tissue by phage buffer. The combined samples, referred to as "non-adherent" cells, are serially diluted, and aliquots of 15 100 μΐ are plated to determine the surviving bacteria.

Example 10

Nisin-EDTA and methylparaben activity against gram-negative bacteria (S. typhimurium)

A bactericide containing nisin and EDTA, combined with a known food preservative, methylparaben, has been shown to be exceptionally effective against gram-negative bacteria, cf. Table X.

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Chicken muscle cubes are rinsed with sodium hypochlorite and povidone iodine prior to use. For grafting the tissue, dice of chicken muscle are dipped into a cell suspension with 108 cells of S. typhimurium per ml. ml in 50 mM 5 sodium acetate buffer having a pH of 5.0. Excess moisture is removed from the dipped cubes upon drainage. The tissue is placed in sufficient buffer containing nisin preparations to cover the tissue and incubated for 2 hours at 37 ° C, then the tissue is transferred to a sufficient amount of 10 phage buffer to cover the tissue. The bacteria that remain in the test solution are collected by centrifugation, washed with phage buffer and combined with bacteria washed out of the tissue by phage buffer. The combined samples, termed "non-adherent" cells, are serially diluted and aliquots 15 of 100 μΐ are plated to determine the surviving bacteria.

In experiment # 1, cf. Table X, methylparaben in the presence of 10 mM EDTA is found to be effective against S. typhimurium at only a concentration of 1.0%. In trial # 2, 20 cf. Table X, where there are 300 µg of nisin per however, the efficacy of methylparaben and nisin against S. typhimurium is significantly improved.

The preparations containing nisin and EDTA significantly improve the usefulness of the food preservative methylparaben. Furthermore, the bactericides can lead to significant reductions in concentrations or eliminate the need for these commonly recognized, albeit less desirable, food preservatives such as methyl paraben.

30 32 DK 171069 B1

Example 11

Nisin and chelating agent activity against gram-positive bacteria (Staphylococcus aureus)

The activation of nisin with a chelating agent is 5 pH dependent. The data in Table XI confirm that nisin at a pH of 5.0 is somewhat more bactericidal against S. aureus than nisin at a pH of 8.0. At a pH of 5.0, EDTA does not increase nisin activity against S. aureus, and at concentrations of EDTA higher than 10 mM 10 EDTA, EDTA is inhibitory to the bactericidal activity of nisin. However, the bactericidal activity of nisin activated with EDTA at a pH of 8.0 is significantly greater than the bactericidal activity of nisin alone or in combination with EDTA at a pH of 5.0.

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The bactericidal activity of nisin alone has been reported, cf. the literature guide to Hurst, to be greatest at a pH of 5.0 or less, and the data set out in Table XI support this. On the basis of this information, it is believed that the bactericidal activation of nisin by EDTA against S. aureus will also be greatest at a lower pH. However, as can be seen from Table XI and contrary to expectations, cf. Table VI, EDTA is not observed to increase nisin activity against gram-positive bacteria 10 at a pH of 5.0. However, inhibition of nisin activity at high concentrations of EDTA is still observed at a pH of 5.0. Thus, the activation of nisin with a chelating agent takes place only within a range of chelating agent concentrations, and in the case of 15 gram-positive bacteria it is dependent on the pH, the preferred pH range being at pH values higher than pH. = 5.0.

Example 12 Nisin and chelating agent activity against gram-positive bacteria

The effects of EDTA on the bactericidal activity of nisin at a pH of 8.0 are not limited to S. aureus, which is a significant human pathogen, but is also observed with Streptococcus mutans responsible for dental plaque, cf. Table XIIA, Listeria monocytogenes, a foodborne pathogen, cf. Table XIIB, and in connection with a mixed population of axillary Coryne form bacteria contributing to body odor, cf. Table XIIC.

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Example 13

Rapid bactericidal activity for nisin activated with chelating agent

The bactericide containing nisin with EDTA, 5 is fast acting bactericide, as illustrated by the data presented in Table XIIIA. Suspensions of the gram-positive bacterium S. mutans with concentrations of 107 cells per ml. ml are incubated in 20 mM Tris buffer, pH = 7.3, at 37 ° C with a range of concentrations of nisin activated with 10 1 mM EDTA. The suspensions are incubated for 0.5 to 60 minutes with the bactericides. The bactericidal effectiveness of the bactericides is assessed by determining the percentage survival of the bacteria. When increasing the activity with EDTA, as low as 10 μg of 15 nisin per ml of this preparation capable of reducing the bacterial load by 6 logarithmic units over 1 minute.

Rapid bactericidal activity is a prerequisite for effective disinfection. Thus, the compositions can be predicted to be effective bactericides, in particular as demonstrated herein, as constituents of mouthwash, rinse and cleansers, toothpaste or other similar tooth and mouth preparations active against plaque-forming S. mutans.

The activity of nisin increased by EDTA against gram-negative bacteria after 2-3 hours is demonstrated in Examples 1-7. The rapid bactericidal activity of nisin increased by EDTA is also seen against gram-negative bacteria, and this is illustrated by the data presented in Table XIIIB.

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Example 14

Effect of divalent cations on EDTA increase of nisin activity

Divalent cations bind to EDTA and other chelating agents and should be expected to neutralize the activation of nisin with EDTA. However, as shown in the data in Table XIV, the bactericidal activity of nisin against S. mutans is increased by 1 mM EDTA, even in the presence of 1 mM Ca 2+ ion. Only at a concentration greater than 3 mM is the Ca 2+ ion 10 inhibiting EDTA-activated nisin. This is of particular importance in applications for mouthwashes or rinses where calcium ion concentrations are relevant.

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Example 15

Nisin and surfactant activity against gram-positive bacteria

The bactericidal activity of nisin can also be significantly increased by combination with a surfactant alone. This is best illustrated by a limiting nisin concentration (0.2 μg / ml) as shown in Table XVA. At concentrations up to 0.1%, the food grade monolaurine surfactant has little significant bactericidal activity against Streptococcus agalactiae in the complex medium milk. Nisin at concentrations up to 0.2 µg / ml also showed no significant bactericidal activity in milk. However, the combination of the two agents, 0.1% monolaurin and nisin at a concentration of 0.2 µg / ml, is highly potent against S. agalactiae. This bactericide is more than 100 times more active than would be expected in terms of an additive effect, and 10,000 times more active than each of the components individually. Thus, when the use of nisin is limited through the available activity of this substance, it may be expected that a bactericide containing nisin together with a surfactant will be more useful.

An example of a case where the use of nisin is limited by the available activity of this substance is illustrated by the data given in Table XVB. Although 25 nisin, and in particular the bactericide containing nisin and EDTA, is bactericidal against L. monocytogenes, the data presented in Table XVB show that the available nisin activity against this organism is limited in a compound medium such as milk. However, the bactericide composed of nisin together with the glyceride 30 monooleate is effective in milk against this pathogen found in foods, although the monooleate itself has no bactericidal action against said organism.

43 DK 171069 B1

TABLE XVA

Bactericidal nisin activity against Streptococcus agalactiae in milk at 37 ° C

(Activation of Nisin with Monolaurin) 5--

Nisin Monolaurin (µg / ml) (%) 0 0.01 0.1 10 Percent survival after 2 hours 0 100 100 4.5 0.02 0.02 100 100 0.2 0.2 2.2 0.05 0.0008 15 aInitially viable bacteria count: 6.0 x 107 cfu / ml Incubations are performed in milk at 37 ° C.

TABLE XVB

Bactericidal nisin activity against Listeria monocytogenes in 20 milk at 37 ° C

(Activation of Nisin with Monooleate)

Nisin Monooleate (μg / ml) (%) 25 0 0.1 1.0

Percentage survival after 2 hours 0 100 67 63 100 0.56 10 “3 10“ 4 3 0 - a Initially viable bacterial count: 5.0 x 107 cfu / ml Incubations are performed in milk at 37 ° C.

Claims (12)

A composition, characterized in that it contains a lanthionine-containing bacteriocin in combination with a chelating and / or surfactant. Composition according to claim 1, characterized in that the lanthionine-containing bacteriocin is selected from the group consisting of nisin, subtilin, epidermin, cinnamycin, duramycin, ancovenin and Pep 5. Composition according to claim 1, characterized in that the chelating agent is selected from the group consisting of alkyl diamine tetraacetates, CaEDTA, Na 2 CaEDTA, EGTA and citrate. Composition according to claim 3, characterized in that the alkyl diamine tetraacetate is EDTA and the bacteriocin is nisin. Composition according to claim 1, characterized in that the surfactant is selected from the group consisting of Triton preparations, Tweens, glycerides, fatty acids, emulsifiers, quaternary compounds, amphoteric surfactants and anionic surfactants. Composition according to claim 1, characterized in that it also contains a food preservative. Composition according to claim 1, characterized in that it also contains a carrier. Composition according to claim 7, characterized in that the lanthionine-containing bacteriocin is selected from the group consisting of nisin, subtilin, epidermin, cinnamycin, duramycin, ancovenin and Pep 5, and the chelating agent is selected from the group consisting of alkyl diamine tetraacetates. EGTA and citrate, and are present in such amounts that increase the antibacterial activity of the preparation against at least one of the bacteria selected from the group consisting of Staphylococcus aureus, Streptococcus mutans, Listeria monocytogenes, Strep-tococcus agalactiae, Coryneform bacteria, Salmonella typhi -murium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas DK 171069 B1 aeruginosa, Bacterioides gingivalis and Actinobacillus acti nomycetescomitans. Composition according to claim 8, characterized in that the alkyl diamine tetraacetate is EDTA. Composition according to claim 7, characterized in that the surfactant is selected from the group consisting of Triton preparations, Tweens, glycerides, fatty acids, quaternary compounds, emulsifiers, amphoteric and anionic surfactants and present in an amount 10 sufficient to increase the antibacterial activity of the composition against at least one of the bacteria from the group consisting of gram-negative and gram-positive bacteria. Composition according to claim 8, characterized in that the concentration of nisin is between approx. 0.1 and approx. 300.0 µg / ml, and the concentration of the chelating agent is between ca. 0.1 and approx. 20 mM. A composition according to claim 10, characterized in that the concentration of surfactant is between 0.01 and approx. 1.0%. 25 30 35